Polycystic kidney disease (ADPKD) results from mutations in the human PKD1 and PKD2 genes, which encode the proteins polycystin-1 and -2. The polycystins are thought to act in the primary cilia of the renal epithelium, where they transduce mechanical bending of the cilium by fluid flow into cytoplasmic calcium signals. Cystogenesis is hypothesized to result from the disruption of this signaling pathway. However, the molecular mechanisms by which the polycystins respond to mechanical cues are not well understood. In the nematode C. elegans, the polycystin orthologs LOV-1 and PKD-2 are required for the function of twenty-one male-specific sensory neurons that sense contact with hermaphrodites. The polycystins localize to the primary cilia at the dendritic tips of these cells. As in vertebrates, it is thought that these proteins transduce the deformation of cilia into downstream calcium signals. Because of its sophisticated and rapid experimental accessibility, the nematode model has great potential for exploring the molecular nature of polycystin signaling. Our goal in this application is to develop and implement a system to directly measure polycystin-dependent calcium signaling in vivo using the genetically-encoded calcium indicator cameleon. In the first aim, we will measure calcium transients in C. elegans males in real time in response to a variety of stimuli, including contact with hermaphrodites, artificial mechanical stimuli, and transverse fluid flow. By comparing calcium responses between wild-type animals and those carrying null mutations in the polycystins, we will test the hypothesis that these responses depend on polycystin function. In the second aim, we will apply this assay to test the hypothesis that two recently-identified C. elegans genes specifically expressed in male sensory neurons, cwp-4 and cwp-5, have roles in polycystin-mediated Ca2+ signaling. These studies will establish C. elegans as a unique system for the in vivo genetic and molecular analysis of the molecular mechanisms of signaling by the polycystins in response to mechanical stimuli. [unreadable] [unreadable] [unreadable]